Glucose can acutely reduce mitochondrial glutathione redox state in rat islets. However, whether glucose-stimulated mitochondrial glutathione redox state relates to glucose-stimulated insulin secretion (GSIS) remains unknown. We used genetically encoded redox-sensitive GFPs to target the mitochondria to monitor glutathione redox changes during GSIS in rat pancreatic beta-cells. The results showed that mitochondrial glutathione was more reduced during GSIS, whereas inhibition of this glutathione reduction impaired insulin secretion. In isolated rat pancreatic islets glutathione reduction in mitochondria and the first phase of GSIS were concurrence at the early stage of glucose-stimulation. Our results suggest that the glucose-induced glutathione reduction in mitochondria is primarily required for the first phase of GSIS. (C) 2015 The Authors. Published by Elsevier Inc.